Despite having evolved from organisms adapted to massive irradiation during the early development of Earth's biosphere, the human body—with its abundance of water—is vulnerable to radiolysis by high-energy (ionizing) irradiation. Medical applications of irradiation critically consider this sensitivity of normal tissues, particularly in the use of total body exposure for bone marrow transplantation patients. However, in uncontrolled situations of exposure to radiation, such as during a terrorist attack, or the unavoidable radiation exposure of flight crews during extended space missions, the development of protective measures is lagging behind, and there is an immediate need for the stockpiling of safe and effective radioprotectors/radiomitigators.
Acute radiation syndrome is associated with damage to the haematopoietic system and gastrointestinal tract due to massive cell loss in radiosensitive tissues occurring largely via apoptosis1,2,3. Along with radicals generated by radiolysis of water, the execution of mitochondria-mediated apoptosis is universally associated with the production of reactive oxygen species (ROS)4,5. Therefore, development of radiomitigators/radioprotectors for biodefense applications and radiotherapy has mostly focused on nonspecific thiol-based antioxidants that have shown clinically insignificant results6,7,8.
Recently, ROS production has been identified as a required step in selective peroxidation of a mitochondria-specific phospholipid, cardiolipin (CL), whose oxidation products are essential for the outer membrane permeabilization and release of pro-apoptotic factors4,9. The catalyst of the peroxidation reaction is cytochrome c (cyt c) that forms a high-affinity complex with CL exhibiting potent peroxidase activity towards polyunsaturated CLs9.
In normal mitochondria, CL and cyt c are spatially separated; the former is confined almost exclusively to the inner mitochondrial membrane, whereas the latter is located in the intermembrane space10. Early in apoptosis, CL migrates from the inner to the outer mitochondrial membrane—a process likely facilitated by one of the four candidate mitochondrial proteins: scramblase-3, nucleoside diphosphate kinase (NDPK-D), mitochondrial isoforms of creatine kinase (m-CPK) and t-Bid11,12,13. Trans-membrane re-distribution of CL makes physical interaction of CL and cyt c possible resulting in the formation of cyt c/CL complexes9.
Several previous studies have proposed that there are two types of interaction of cyt c with anionic phospholipids; an electrostatic interaction and a specific hydrophobic interaction. Whereas the electrostatic interaction is mainly driven by the charges between the protein and anionic lipids, the hydrophobic interaction involves the insertion of the lipid acyl chain in a hydrophobic channel present in the structure of cyt c. It has been shown that both interactions are essential for initiating the peroxidase activity of cyt c9 leading to peroxidation of bound polyunsaturated molecular species of CL. Notably, accumulation of peroxidized CL is essential for the execution of the apoptotic program. Conversely, prevention of CL peroxidation leads to inhibition of apoptosis14.
Cyt-c-mediated apoptosis has also been associated with apoptosis in freeze-thaw treatment of cells (i.e. cryopreservation).15 
Also, in instances of ischemic stroke, the border region of less severely affected brain tissue that surrounds the core necrotic area remains metabolically active following the ischemic event16. Many neurons in this region undergo apoptosis in the hours or days following the stroke event, thus there is potential to save such neurons through some form of post-stroke therapy that can suppress apoptosis.16 Myocardial infarctions are another type of ischemic event that results in a similar manner of cell death.
Ischemia is pathological condition in which blood flow to cells of an organ or tissue is restricted for a period of time, subsequently followed by restoral of perfusion and associated reoxygenation.17 During ischemia, the reduction in blood flow limits the supply of oxygen and glucose to cells and gradually leads to hypoxia/anoxia, resulting in an ultimate disruption of cellular homeostasis.17 Re-establishment of blood flow restores oxygen and nutrient supply to cells, but causes further damage to cells.17 For example, the burst of oxygen to cells after blood supply has been restored can cause an increased production of harmful reactive oxygen species (ROS) within mitochondria that may oxidize and damage macromolecules within the cell.18 As a result of Ischemia/Reperfusion (I/R) injury, cell death occurs.17 
Apoptosis is among the modes of cell death that occur in response to I/R.17 Mitochondrial events appear to have important contributions to the apoptotic pathway associated with many kinds of I/R events, such as cerebral I/R and myocardial infarction.19,20 Pro-apoptotic tBid appears to be an early activator of the mitochondrial pathway of apoptosis in both neuronal and focal cerebral I/R.21 Cyt c and Smac/Diablo release from mitochondria, as well as caspase-9 and caspase-3 activation have also been reported to occur in response to cerebral I/R.19 
Oxygen-glucose deprivation (OGD) appears to be an accepted model for in vitro studies of cerebral ischemia that is commonly used. This model was first developed by Goldberg and Choi22, and involves the combined deprivation of oxygen and glucose followed by restoration of normal culture conditions to cultured neural cells roughly approximating the conditions of in vivo cerebral ischemia/reperfusion (I/R). Exposure of neural-like cells to OGD leads to patterns of cell death associated with cerebral ischemia22, and so it can be used for the study of cell death mechanisms associated with cerebral ischemia or the effectiveness of potential protective strategies.
SH-SY5Y cells, a human neuroblastoma cell line, have been frequently used in OGD studies as a model of neuronal cell death associated with cerebral I/R. Fordel et al.23 found that cell viability was 54% after 32 hour recovery from 16 hour OGD exposure, and a pattern of cell death just like that of in vivo cerebral ischemia was observed. That is, necrotic cell death predominantly occurred during OGD, while apoptotic death was greater following restoration of oxygen and glucose23. In another study, Wang et al.24 exposed SH-SY5Y cells to varying times of OGD, and found that a 10-16 hour exposure decreased viability to cells to below 50% after 72 hours of recovery. Serra-Perez et al.25 examined retinoic acid-differentiated SH-SY5Y cells in the context of OGD, and found that cyt c was present in the cytosol after 6 hours of reoxygenation following a 15 hour OGD period. Furthermore, an increase in the amount of apoptotic cells and caspase-3 activity was observed during the recovery period25.
In contrast to the relatively long periods of OGD used in the above studies, Agudo-Lopez et al.26 exposed SH-SY5Y cells to a much shorter period of OGD, lasting 3 hours in duration. After 16 hours of recovery, a 33% decrease in viability compared to cells not exposed to OGD was observed, along with a small percentage of cells at early apoptotic stages.